Atherosclerosis, a disease of arteries that is responsible for most cardiovascular-related morbidity and mortality, develops in predictable regions of the arterial tree that correlate with complex patterns of blood flow. Although many atherosclerotic lesions are mild and cause little harm, those that progressively obstruct the passage of blood may reduce oxygen delivery to levels below the needs of the tissue (e.g., angina pain when coronary arteries are affected) or precipitate an acute ischemia (e.g., heart attack, stroke) when blood clots form on a destabilized lesion surface. Another catastrophic consequence of advanced lesions is the weakening of the artery wall, leading to pressure-induced ballooning (aneurysm) and potential rupture. It has long been recognized that hemodynamics determines the location of lesions. Local vessel geometry (e.g., arterial branching and curvatures), and constraint of vessel motion by surrounding tissues (e.g., coronary arteries) lead to flow instabilities and separations that correlate with sites of lesion development.
The one-cell thick layer at the interface between flowing blood and the artery wall is called the endothelium. Two decades of intense research have shown that the endothelium, rather than being a simple passive barrier, is instead both i) a multifunctional effector of systemic and vessel wall biology, and ii) an exquisitely sensitive responder to the local environment. The endothelium is directly exposed to the hemodynamic shear stresses associated with all of the different flow characteristics found in the circulation.
Endothelial cell responses to the hemodynamic environment are frequently heterogeneous. Prominent examples are the expression of VCAM-protein in vivo (Walpola et al., 1995, Arterioscler. Thromb. Vasc. Biol. 15:2-10) and in vitro (Ohtsuka et al., 1993, Biochem. Biophys. Res. Comm. 193:303-310), VCAM-1 mRNA expression in vivo (McKinsey et al., 1995, FASEB J. A343), ICAM-1 protein expression in vivo (Walpola et al., 1995, Arterioscler. Thromb. Vasc. Biol. 15:2-10) and in vitro (Nagel et al., 1994, J. Clin. Invest. 94:885-891), elevation of intracellular calcium ([Ca2+]I) measured in vitro (Geiger et al., 1992, Am. J. Physiol. 262:C1411-C1417; Shen et al., 1992, Am. J. Physiol. 262:C384-C390) and in vivo (Falcone et al., 1993, Am. J. Physiol. 264:H653-659), induction of synthesis and nuclear localization of c-fos in vitro (Ranjan et al., 1993, Biochem. Biophys. Res. Comm. 196:79-84), expression of major histocompatibility complex (MHC) antigens in vitro (Martin-Mondiere et al., 1989, ASAIO Trans. 35:288-290), inhibition of endothelial cell division in vitro (Ziegler et al., 1994, Arterioscler. Thromb. 14:636-643), and re-localization of the Golgi apparatus and microtubule organizing center (MTOC) in vitro (Coan et al., 1993, J. Cell Sci. 104:1145-1153). In each of these cases, high levels of response in one cell or a group of cells are accompanied by absent or diminished responses in adjacent cells of the same endothelial monolayer despite exposure to a substantially identical bulk flow field in vitro, or location in a predicted uniform hemodynamic environment in vivo.
In vitro, nominal flow characteristics are defined by the geometry of the experimental system (e.g., flow tube, parallel plate, cone and plate, etc.). The average wall shear stress and shear stress gradient values can be accurately estimated or directly measured (Dewey et al., 1981, J. Biomech. Eng. 103:177-188; Davies et al., 1986, Proc. Nat. Acad. Sci. USA 83:2114-2118; Olesen et al., 1988, Nature 331:168-170; DePaola et al., 1992, Arterioscler. Thromb. 12:1254-1257). Although the flow characteristics are more complex in vivo, average shear stress values can be estimated from vessel geometry and flow rates (Zarins et al., 1983, Circ. Res. 53:502-514). Such measurements demonstrate that although all of the cells in a given region of the monolayer are estimated to be subject to very similar shear stresses calculated from bulk flow characteristics, there are substantial cell-to-cell differences in acute and chronic responses to flow. If, as a significant number of experiments demonstrate, the responses are related to hemodynamic forces, it has not been determined what accounts for the heterogeneous responses.
In vitro flow chamber models of disturbed and undisturbed blood flow as described herein have recently been used to identify regionally defined differential expression of connexin43, and early response genes (DePaola et al., 1999, Proc. Natl. Acad. Sci. USA, 96:3154-3159; Nagel et al., Arterioscler. Thromb. Vasc. Biol., In press.) In regional differential gene expression studies during flow in vitro, endothelial cells are typically isolated by scraping the regions of interest. If enough cells are recovered, quantitative estimates of regional up- or down-regulation of gene expression (i.e., an average from all of the cells isolated from a particular location) can be made by northern blot analyses using specific nucleic acid probes for each gene of interest.
A useful alternative for analyzing the smaller numbers of cells typically present in defined hemodynamic regions is differential-display PCR (ddPCR; Liang et al., 1992, Science, 257:967-971), which uses reverse-transcription PCR (RT-PCR) to amplify all expressing genes in the cell population. This allows evaluation of differential expression of multiple genes when PCR products derived from cells in different hemodynamic regions are displayed together (e.g., as in Topper et al., 1996, Proc. Natl. Acad. Sci. USA, 93:10417-10422). Although ddPCR can be imprecise for quantitation of expression, this method has been used to identify differentially-expressed genes (e.g., Topper et al., 1997, Proc. Natl. Acad. Sci. USA, 94:9314-9319; Topper et al., 1997, J. Clin. Invest., 99:2942-2949; Topper et al., 1997, Proc. Natl. Acad. Sci. USA, 94, 9314-9319).
Although regional differential gene expression studies as described above are of value, the hemodynamic effects which modulate endothelial gene expression through spatial and temporal shear-stress relationships are ultimately defined locally at the surface of individual endothelial cells. Surface topographies, and consequently the magnitudes and gradients of shear-stresses, vary considerably from cell to cell (Barbee et al., 1995, Am. J. Physiol., 268:H1765-H1772). Differences in hemodynamic signaling and gene expression that have been observed from region to region and from cell to cell in endothelium (both in culture and in tissues) are likely to arise from microscopic topographic differences at the interface of the fluid and the cell surface. Examples of such heterogeneity include variable expression of endothelial vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 proteins from cell to cell in vivo (Walpola et al., 1995, Arterioscler. Thromb. Vasc. Biol., 15:2-10 ; Nakashima et al., 1998, Arterioscler. Thromb. Vasc. Biol., 18:842-851) and in vitro (Nagel et al., 1994, J. Clin. Invest., 94:885-91), the elevation of intracellular calcium measured in vitro (Geiger et al., 1992, Am. J. Physiol., 262:C1411-1417; Shen et al., 1992, Am. J. Physiol., 262:C384-C390) and in vivo (Falcone et al., 1993 Am. J. Physiol., 264:H653-H659), the induction of synthesis and nuclear localization of c-Fos in vitro (Ranjan et al., 1993, Biochem, Biophys. Res. Commun., 196:79-84), and the expression of major histocompatibility complex antigens in vitro (Martin-Mondiere et al., 1989, ASAIO Trans., 35:288-290). In all of these studies, highly variable responses were observed in adjacent cells of the same endothelial monolayer exposed to a nominally identical flow field.
Two inter-related mechanisms may explain cell-to-cell differences in acute and chronic responses to hydrodynamic forces. First, there may be differential expression or sensitivity of mechano-sensing or transduction systems in the endothelial cells, ranging from a complete absence to super-sensitivity. Second, the shear stresses and shear stress gradients acting on the cells may be heterogeneous because of differences in the detailed cell surface topography. Atomic force microscopy (AFM) and computational fluid dynamics (CFD) have been used to detail the geometry of living endothelial cell surfaces in vitro and in situ and to calculate the sub-cellular localized force distribution (Barbee et al., 1995, Am. J. Physiol. 268:H1765-H1772; Davies, 1995, Physiol. Rev. 75:519-560; Davies et al., 1997, Ann. Rev. Physiol. 59:527-549). Results of these studies indicated that microscopic hydrodynamic forces acting on individual endothelial cells vary considerably from cell to cell and within different regions of a single cell. It appears that, both in vivo and in vitro, differential responsiveness to macroscopically uniform shear stresses occurs because of microscopic heterogeneities. These studies assume importance in view of the complex flow fields associated with atherogenesis, and address the fundamental basis of atherosclerotic focal origin, lesion initiation, and progression.
An important implication of these findings is that expression of a limited number of genes in even only a few endothelial cells can dominate vascular physiology and vascular pathogenesis. However, the identity of such genes can be complicated by xe2x80x9cdilutionxe2x80x9d of mRNA transcribed from such genes by more numerous mRNA species, particularly when the pool of cells from which mRNA is isolated includes only a few xe2x80x9cdominantxe2x80x9d cells.
Advances in single cell and mRNA amplification techniques and methods of obtaining quantitative profiles of gene expression (e.g., transcriptional profiles) including the use of gene arrays for high throughput analyses now allow one to address endothelial heterogeneity in a very detailed (e.g., single cell and small groups of cells) yet comprehensive (multiple genes, high-throughput) approach. However, little is currently known about the genes involved in mediating the heterogeneous responses of endothelial cells to hemodynamic forces. Thus, there is an unmet need in the art for methods and compositions related to focal hydrodynamic stress-related regulation of gene expression which are useful in the development of methods for the prevention and treatment of atherosclerosis and other cardiovascular diseases in humans. The present invention satisfies those needs by providing the means to characterize the gene expression profile(s) of a single cell or small groups of cells present in a local hemodynamic environment that promotes susceptibility to atherosclerosis in vivo, or simulates in vitro the flow disturbances associated with atherosclerosis.
The invention includes a method of identifying a gene, expression of which is regulated by hydrodynamic stress. The method comprises a) subjecting a first vascular endothelial cell of a mammal to hydrodynamic stress; b) thereafter assessing the level of expression of the gene in the cell, and c) comparing the level of expression of the gene in the first cell with the level of expression of the same gene in a second vascular endothelial cell of the mammal, the second cell being subjected to different hydrodynamic stress, whereby a difference between the level of expression of the gene in the first cell and the level of expression of the gene in the second cell is an indication that the gene is regulated by hydrodynamic stress.
In one aspect, the cell is subjected to hydrodynamic stress using an in vitro flow chamber.
In a preferred embodiment, the flow chamber is capable of generating a hydrodynamic stress flow field having spatially defined microheterogeneity.
In another aspect, the microheterogeneity results in different hydrodynamic stresses exerted from one cell to another or from one part of a cell to another part of a cell in the flow chamber.
In one embodiment, the mammal is a human.
In one aspect, hydrodynamic stress is exerted in an amount from about 0 dyn/cm2 to about 100 dyn/cm2.
In another aspect, the hydrodynamic stress is exerted for at least about several seconds.
In one embodiment, the cell is a single cell isolated in vivo.
In a preferred embodiment, the cell is an arterial endothelial cell.
In another embodiment, the cell is a group of cells isolated in vivo.
In yet another embodiment, the cell is a single cell isolated in vitro from a confluent monolayer.
In one aspect, the cell is one of a group of cells isolated in vitro from a confluent monolayer.
In a preferred embodiment, the level of expression of the gene is assessed using amplified antisense RNA in combination with northern blotting or a microarray technique.
In one aspect, the level of expression of the gene is assessed in a single endothelial cell.
In another aspect, the level of expression of the gene is assessed in one of a group of endothelial cells.
In yet another aspect, the level of expression of the gene is compared by transcriptional profiling following one of northern blotting and microarray analysis using one of a radiolabeled probe, a fluorescent probe and a label.
In a further aspect, the levels of expression of a plurality of genes are compared. The invention also includes a method of identifying a nucleic acid comprising a hydrodynamic stress regulation (HSR) region. The method comprises
a) subjecting a first vascular endothelial cell of a mammal to hydrodynamic stress;
b) thereafter assessing the level of expression of the nucleic acid in the cell, and
c) comparing the level of expression of the nucleic acid in the first cell with the level of expression of the same nucleic acid in a second vascular endothelial cell of the mammal, the second cell being subjected to different hydrodynamic stress, whereby a difference between the level of expression of the nucleic acid in the first cell and the level of expression of the nucleic acid in the second cell is an indication that the nucleic acid comprises a HSR region.
In one embodiment, the cell is subjected to hydrodynamic stress using an in vitro flow chamber capable of generating a hydrodynamic stress flow field having spatially defined microheterogeneity.
In another embodiment, the level of expression of the nucleic acid is assessed using amplified antisense RNA from a single endothelial cell.
Also included in the invention is a method of identifying a nucleic acid comprising an HSR region. The method comprises a) comparing the sequence of a first nucleic acid with the sequence of a second nucleic acid comprising an HSR region, and b) identifying a region of the first nucleic acid which is homologous to the HSR region of the second nucleic acid, whereby a nucleic acid comprising a HSR region is identified.
Furthermore, the invention includes a method of identifying a hydrodynamic stress responsive protein. The method comprises a) subjecting a first vascular endothelial cell of a mammal to hydrodynamic stress; b) thereafter assessing the level of expression of a nucleic acid in the first cell, and c) comparing the level of expression of the nucleic acid in the first cell with the level of expression of the same nucleic acid in a second vascular endothelial cell of the mammal, the second cell being subjected to different hydrodynamic stress, whereby a difference between the level of expression of the nucleic acid in the first cell and the level of expression of the nucleic acid in the second cell is an indication that the nucleic acid encodes a hydrodynamic stress responsive protein, and d) identifying a protein encoded by the nucleic acid, whereby a hydrodynamic stress responsive protein is identified.
In one embodiment, the cell is subjected to hydrodynamic stress using an in vitro flow chamber capable of generating a hydrodynamic stress flow field having spatially defined microheterogeneity.
In another embodiment, the level of expression of the nucleic acid is assessed in a single endothelial cell.
The invention also includes an array of nucleic acids comprising an HSR region, wherein at least one of the nucleic acids is selected by a method of the invention.
Additionally, the invention includes, a kit for carrying out a method of the invention. The kit comprises a) an instructional material; b) a reagent for use in amplified antisense RNA; c) a reagent for use in northern blotting or microarray analysis, and d) a radiolabeled or fluorescent probe.